Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod (G. morhua) using a 96-well microplate technique

Zakhartsev, Maksim; Pörtner, Hans‐Otto; Blust, Ronny

doi:10.1016/j.ab.2004.03.070

Cited by 8 publications

(4 citation statements)

References 23 publications

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“…Several suggestions have been made to overcome edge effects, mostly in a specific constructive way changing the incubation chambers or plates . Commercial MTP readers usually apply external heating units with air as heat transfer medium and feature typically ±0.2 to 0.5°C accuracy according to manufacturer's specifications.…”

Section: Introductionmentioning

confidence: 99%

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Grosch

Sieben

Lattermann

et al. 2016

Biotechnology Journal

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Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.

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Section: Introductionmentioning

confidence: 99%

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Grosch

Sieben

Lattermann

et al. 2016

Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…When transferring cuvette‐based enzymatic assays to MTPs, the deviation is often so high that the experimental setup needs independent optimization . The reproduction of kinetic data is even more challenging for MTPs or heating chambers that have been developed to overcome issues as irregular heating or meniscus formation . Our investigations show that reliable kinetic data are highly dependent on the experimental setup used and highlight the importance to properly state the experimental.…”

Section: Resultsmentioning

confidence: 94%

“…[12][13][14]32 The reproduction of kinetic data is even more challenging for MTPs or heating chambers that have been developed to overcome issues as irregular heating or meniscus formation. 4,[33][34][35][36] Our investigations show that reliable kinetic data are highly dependent on the experimental setup used and highlight the importance to properly state the experimental.…”

Section: Qualitative Comparison Of Experimental Systems and Error Quamentioning

confidence: 99%

Influence of the experimental setup on the determination of enzyme kinetic parameters

Grosch

Wagner

Knaup

et al. 2016

Biotechnology Progress

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For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29-118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87-95, 2017.

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“…Repetitive batch cultivations at varied temperatures, either in shake flasks or bench scale bioreactors, provide only a few data points at the expense of relatively high material and time input. The analysis of temperature-specific activities and kinetic parameters of enzymes is traditionally performed in a spectrophotometric way by use of a temperature-controlled water jacketed single cuvettes [5]. Even though the external thermostat provides constant and accurate temperature control over a broad temperature range, the numbers of samples handled for simultaneous reading in such spectrophotometers are usually very limited.…”

Section: Introductionmentioning

confidence: 99%

Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

et al. 2014

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BackgroundBioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.ResultsIn this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.ConclusionMicrotiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

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Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod (G. morhua) using a 96-well microplate technique

Cited by 8 publications

References 23 publications

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers

Influence of the experimental setup on the determination of enzyme kinetic parameters

Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

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